Mycoplasma fermentans, a species that colonizes humans, is unique among mycoplasmas. Most mycoplasmas are divided metabolically into species that ferment glucose or species that are nonfermenters but hydrolyze arginine. M. fermentans, however, possesses both metabolic pathways. Furthermore, M. fermentans, apparently unlike other human mycoplasmas which are obligate extracellular parasites, is an intracellular parasite that has been implicated as the agent of a fatal necrotic disease. The arginine dihydrolase pathway includes arginine deiminase (AD), which converts L-arginine to L-citrulline; ornithine transcarbamylase (OTC) which converts citrulline to ornithine and carbamyl phosphate; and carbamate kinase, which yields carbamyl phosphate to CO2, ATP and NH3. AD is an immunosuppressive substance which has been shown to exert a lymphocyte blastogenesis inhibitory activity. If M. fermentans is capable of causing an immunosuppressive disease, better understanding of its metabolism is warranted. Since both fermentation and arginine hydrolysis are occurring within the cell, we investigated whether repression by glucose of arginine hydrolysis is a regulatory mechanism used by M. fermentans. Whole cell perfusion NMR was used to demonstrate that M. fermentans is ferments glucose concomittantly with arginine hydrolysis. Enzyme assays of the AD from M. fermentans grown with or without glucose did not show any repression of the activity. In contrast, the specific acitivities of M. fermentans OTC from cells grown with or without glucose demonstrated vividly that this enzyme was subject to glucose repression. The OTC from M. hominis was, as expected, unaffected by growth in glucose. Therefore regulation of the arginine dihydrolase pathway in M. fermentans occurs in large part at the level of OTC repression by glucose, while arginine deiminase is unaffected by glucose metabolism.